We have previously tested immunotherapeutic and immunoprophylactic recombinant glycoprotein herpes simplex virus type 2 (HSV-2) vaccines in humans; unfortunately, these studies were unsuccessful in phase III clinical trials. Thus, a more effective HSV-2 vaccine is needed. We have concentrated our efforts on other HSV-2 vaccines, and on animal models to try to determine the immunologic and viral factors that correlate with latency and reactivation.[unreadable] [unreadable] We have postulated that the limited efficacy of HSV-2 recombinant glycoprotein D (gD) vaccines is likely due to inadequate induction of cellular immune responses. We have evaluated a candidate HSV-2 vaccine deleted for two essential genes, termed HSV-2 dl5-29, which was developed by David Knipe at Harvard University. Previously, we compared the cellular immune response of HSV-2 dl5-29 to recombinant HSV-2 gD in mice and found that HSV-2 dl5-29 resulted in significantly higher systemic HSV-2 specific lymphocytes (CD8 T cells) responses than gD. HSV-2 dl5-29 also induced more rapid accumulation of HSV-2 specific lymphocytes (CD8 T cells) in trigeminal ganglia after challenge with wild-type virus compared to HSV-2 gD. In addition, we evaluated the vaccine in female guinea pigs in which infection with HSV-2 results in spontaneous reactivations. HSV-2 dl5-29 prevented acute, recurrent, and latent infection in guinea pigs as effectively as HSV-2 recombinant gD. HSV-2 dl5-29 was more effective than recombinant HSV-2 gD when the vaccine was given immunotherapeutically. When guinea pigs that were previously infected with wild-type HSV-2 were inoculated with HSV-2 dl5-29 or recombinant gD, animals given HSV-2 dl5-29 had lower recurrence rates compared to those receiving HSV-2 gD. Guinea pigs vaccinated with HSV-2 dl5-29 had higher levels of virus neutralizing antibodies in the sera compared with animals inoculated with recombinant HSV-2 gD. Since the cellular immune and neutralizing antibody responses to HSV-2 dl5-29 were superior to that seen with recombinant HSV-2 gD, HSV-2 dl5-29 is an attractive candidate for early phase clinical trials in humans. [unreadable] [unreadable] We are currently testing a derivative of HSV-2 dl5-29 in which an additional HSV-1 gene has been deleted from this virus. The deleted gene normally impairs the immune respopnse to the virus; therefore animals infected with the deletion mutant may have improved immune responses and protection after challenge virus with wild-type virus. [unreadable] [unreadable] We are also studying the contribution of (a) the number of infiltrating virus-specific lymphocytes (CD8 T cells), and (b) the latent viral load, to the rate of reactivation of HSV-2-infected neurons ex vivo. While the rate of HSV reactivation correlates to some extent with the latent viral load, the latent viral load does not fully account for the rate of reactivation, and other factors contribute to reactivation. We postulate that cellular immune responses might also influence reactivation. Therefore we have inoculated mice with HSV-2, dissociated trigeminal ganglia and measured (a) numbers of infiltrating lymphocytes (CD8 T cells) by flow cytometry, (b) the latent viral load by real time PCR, and (c) the rate of reactivation by quantifying the amount of cell-free HSV released into the media of the cultures. These studies should indicate the relative role of certain lymphoyctes (CD8 T cells) in controlling reactivation rates.